Intravascular fibrin deposits are a common feature of many disorders, yet the underlying molecular mechanisms remain obscure. The overall goal of this proposal is to delineate the changes in hemostatic molecules which lead to fibrin deposition in vascular disease. The primary hypothesis is that genes that control the initiation of the coagulation (i.e., tissue factor, TF; TF pathway inhibitor, TFPI) and fibrinolytic (i.e., tissue plasminogen activator, t-PA; urokinase PA, u-PA; u-PA receptor, u-PAR; and PA inhibitor 1, PAI-1) cascades are normally expressed by a variety of tissues in vivo, and their relative level of expression is altered in specific tissues in specific disorders, sometimes resulting in pathological thrombosis. A second hypothesis is that the failure to detect fibrin in some tissues reflects a hyperactive fibrinolytic system in those tissues, and that u-PA plays a fundamental role in this process. Each specific aim employs a different murine model. PAI-1, t-PA or u-PA deficient mice are used to test the generality of these hypotheses, and to identify the cells and clarify the mechanisms that control their expression in vivo. Competitive RT-PCR, in situ hybridization immunoassays and immuno-histochemistry will be used both to quantitate and localize these mRNAs and proteins in tissues and cells from endotoxin-treated mice (Aim 1) and from mice that develop glomerulonephritis (GN; Aim 2), obesity (Aim 3) and atherosclerosis (Aim 4), and to relate them to fibrin deposition. The potential role of u-PA in fibrinolysis will be tested in endotoxin experiments using u-PA deficient mice, by pre-treating mice with tranexamic acid prior to endotoxin, and by crossing GN mice with u-PA deficient mice. The hypothesis that elevated PAI-1 promotes fibrin in GN, obesity and atherosclerosis will be addressed by crossing diseased mice with PAI-1 deficient mice. Finally, genetically obese mice will be employed to test the hypotheses that the adipose tissue is the major source of elevated plasma PAI-1, and that this increase reflects increased synthesis in response to chronically elevated levels of tumor necrosis factor alpha and insulin. These studies will provide insights into the regulation of these genes in vivo, should identify factors that control pathological fibrin-deposition in tissues, and may ultimately lead to the development of strategies to treat these disorders.